Disease-modifying pharmacological treatments of type 1 diabetes: Molecular mechanisms, target checkpoints, and possible combinatorial treatments

Abstract

After a century of extensive scientific investigations, there is still no curative or disease-modifying treatment available that can provide long-lasting remission for patients diagnosed with type 1 diabetes (T1D). Although T1D has historically been regarded as a classic autoimmune disorder targeting and destroying pancreatic islet β-cells, significant research has recently demonstrated that β-cells themselves also play a substantial role in the disease's progression, which could explain some of the unfavorable clinical outcomes. We offer a thorough review of scientific and clinical insights pertaining to molecular mechanisms behind pathogenesis and the different therapeutic interventions in T1D covering over 20 possible pharmaceutical intervention treatments. The interventions are categorized as immune therapies, treatments targeting islet endocrine dysfunctions, medications with dual modes of action in immune and islet endocrine cells, and combination treatments with a broader spectrum of activity. We suggest that these collective findings can provide a valuable platform to discover new combinatorial synergies in search of the curative disease-modifying intervention for T1D. SIGNIFICANCE STATEMENT: This research delves into the underlying causes of T1D and identifies critical mechanisms governing β-cell function in both healthy and diseased states. Thus, we identify specific pathways that could be manipulated by existing or new pharmacological interventions. These interventions fall into several categories: (1) immunomodifying therapies individually targeting immune cell processes, (2) interventions targeting β-cells, (3) compounds that act simultaneously on both immune cell and β-cell pathways, and (4) combinations of compounds simultaneously targeting immune and β-cell pathways.

Fig. 1

β-cell regulation of electrical activity and insulin secretion. β-cell function, Ca²⁺ management, and insulin secretion are regulated by the coordinated interaction of ionic movements. (0) In the absence of glucose, the K_ATP channel is open, maintaining the β-cell membrane hyperpolarized and electrically silent. (1) Glucose influx initiates electrical activity through an increase in metabolically produced ATP, (2) which binds to K_ATP and gradually reduces its conductance, causing an increase in the β-cell membrane potential. (3) β-cell is slowly depolarized in waves, activating voltage-gated calcium (VGCC) and sodium (VGSC) channels and eliciting exocytosis of insulin. (4) Continued depolarization subsequently activates voltage-gated potassium channels (K_V). (5) K⁺ efflux initiates and regulates the repolarization and consequent hyperpolarization of the cell membrane action potential. ATP-dependent Na⁺/K⁺-ATPase (NKAs) activate by multiple metabolic factors, including insulin receptors and once activated (6) further hyperpolarize β-cell membrane, temporarily halting β-cell electrical excitability, closing K_V and inhibiting influx of Na⁺ and Ca²⁺ entry, thereby reducing insulin secretion. (7) G_i/o-GPCR ligands signaling also activates NKAs and (8) hyperpolarizes β-cell membrane halting Ca²⁺ influx and insulin secretion. Reduction in ATP during hyperpolarization allows a new increase in β-cell membrane potential, leading to another wave of depolarization and insulin secretion. (9) The GSIS is potentiated by G_S-GPCR stimulation with GLP-1, GIP, and other G_S-ligands that activate adenylyl cyclase (AC) thereby elevating levels of intracellular cAMP. cAMP drives changes including Ca²⁺ influx, mobilization, and the enhanced fusion competence of secretory granules. (10) Elevation of intracellular cAMP concentrations and the activation of cAMP-dependent protein kinase A (PKA) leads to the opening of IP₃ receptors in ER, releasing Ca²⁺ from internal stores, notably the ER, thereby potentiating GSIS and promoting insulin exocytosis even in Ca²⁺ depleted conditions. (11) PKA activity in β-cell will also inhibit NKA function and promote β-cell depolarization. (12) Independently of PKA, cAMP also activates Epac2-mediated engagement of Rap1 in β-cells that increase the size of the nondocked granule pool and facilitate recruitment and density of the granules to the plasma membrane, thereby potentiating insulin secretion even at low concentrations of glucose. (13) Depolarization independent from glucose influx can occur when β-cell is exposed to GABA, through GABA_A receptor (GABA_AR) opening allowing for Cl^- efflux. GABA is synthesized in β-cells from glutamate with the GAD65 enzyme and can activate GABA receptors on the β-cell in a positive feedback loop. GAD65 is distributed between ER/Golgi membrane-anchored, vesicular, and cytosolic localizations. Although a minority of β-cells display vesicular GABA colocalization with insulin, most β-cells release GABA via volume regulatory anion channels (VRAC) in a pulsatile pattern, occurring in rhythmic bursts independent of glucose concentration. (14) Insulin expression in β-cells is achieved by a glucose-dependent transcriptional program. Three transcriptional regulators, PDX1, NeuroD1, and MafA are responsible for glucose-induced insulin gene transcription and β-cell-specific function. (15) Insulin gene encodes mRNA preproinsulin, which is translated and translocated across the ER membrane where the signal peptide is removed. The resulting proinsulin molecules are subsequently folded and transported to the Golgi apparatus via the ER-Golgi interface compartment. (16) Upon exposure to acidity and high Ca²⁺ within the Golgi apparatus, the soluble cargo precursor proteins aggregate and bind Ca²⁺, which triggers the aggregation of proinsulin molecules in the trans-Golgi network (TGN). Insulin secretory granule (SG) cargo, including proinsulin, is packaged into granules that bud off from the TGN. (17) SG matures by acidification through the action of the vesicular ATP-dependent proton pump (V-ATPase). Proton (H⁺) transport over the granular membrane results in the development of a considerable membrane potential (ΔΨ) and pH changes. Uptake of negatively charged glutamate (Glu-) by VGLUT3 sets up a counter-charge movement which decreases the granular membrane polarization allowing sustained H⁺ transport by the V-ATPase. EAAT2 in the SG provides a mechanism for the release of accumulated Glu^-, 3Na⁺, and an H⁺ exchanged with 1K⁺, which decreases the granular membrane polarization allowing sustained acidification by the V-ATPase. Cl^- fluxes through the CLC3 channel in the SG membrane provide additional counterconductance for continuous granular acidification. Acidification lowers the pH in the granule and activates prohormone-processing enzymes PC1/3, PC2, and CPE that convert proinsulin to active insulin inside the SG. (18) Insulin exocytosis occurs through a process involving SG docking to the plasma membrane, followed by priming and inward Ca²⁺ and Na⁺ flux-dependent release.

Fig. 2

Proposed chart of T1D dual vulnerability initiated by β-cell metabolic dysfunction and CD4+ T-cell activation. β-cell metabolic dysfunction processes (orange numbers). (1) Chronic exposure to hyperglycemia causes elevation of the carbohydrate response-element binding protein β (ChREBPβ) and Ca2+ influx, leading to increased transcription of proapoptotic TxNIP. (2) In the β-cell TxNIP upregulates transcription of NLRP3 inflammasome, a protein complex responsible for caspase-1–dependent maturation of the proinflammatory cytokines IL-1β and IL-18 and gasdermin D(GSDMD)-mediated apoptotic cell death. (3) The onset of metabolic dysfunction may also arise from the impaired glutamate transmission and dysregulation of the β-cell’s N-methyl-d-aspartate receptors (NMDARs), which play a crucial role in controlling insulin secretion, electrical activity, and cell survival by modulating the influx of calcium ions (Ca2+) and sodium ions (Na+). Increases in cytosolic Ca2+ levels cause increased permeability of mitochondria, altered mitochondrial respiration, the release of reactive oxygen species (ROS), and activation of other proapoptotic factors. Breakdown of the depolarization mechanisms precludes repolarization via the opening of voltage-gated K+ channels (KV), which can have an excitotoxic effect on β-cells and increase intracellular Ca2+ levels. (4) β-cell metabolic dysfunction could suppress GABA synthesis from intracellular glutamate by glutamic acid decarboxylase (GAD65) and release of GABA from β-cells. GAD65 is one of the major target antigens in T1D, and GAD65 autoantibody is a diagnostic marker for T1D. Patients with T1D exhibit a significant reduction in plasma GABA levels. This impacts islet regulatory pathways, including a key β-cell mechanism for glucagon regulation in the α-cells via chloride (Cl-) influx through GABAAR and hyperpolarization of the α-cell plasma membrane. Insufficient extracellular GABA levels also hinder cAMP-dependent β-cell survival pathways, such as β-catenin-mediated (β-cat) signaling and GABAB receptor (GABABR) initiated PI3K-AKT-cascades. Additionally, reduced GABA signaling will also fail to inhibit T cell proliferation through activating GABAA Cl- channels and reducing the secretion of interferon gamma, IL-6, IL-12, IL-1β, and TNF-α. (5) Cellular stress includes endoplasmic reticulum (ER) stress which triggers an unfolded protein response (UPR) consisting of impaired RNA transcription and translation, leading to depletion of ER Ca2+ stores and accumulation and release of misfolded proteins, hybrid insulin peptides (HIP) and increased autoantigen presentation. (6) Elevated serum DPP-4 (sDPP-4) levels are found to be elevated in T1D patients and degrade incretins such as GLP-1, thereby preventing incretin-induced cAMP downstream signaling in β-cells, which interferes with GSIS and impairs cAMP-dependent cell survival processes. (7) An inflammatory trigger event in β-cells (eg, viral infection via coxsackie and adenovirus receptor [CAR]) would initiate secretion of type I interferons (interferon-α/β) by the immune system or other cells, leading to activation of the JAK-STAT pathway and the NF-κB pathway and increase of ER stress in the β-cell. (8) interferon alfa has been identified as a key driver of increased expression of HLA class I molecules of the major histocompatibility complex I (MHC1) system on β-cells in the early stages of T1D. These MHC1 molecules bind β-cell–derived autoantigens (β-Ag) and activate CD8+ T-cells, as well as upregulate ER stress sensors and markers in the β-cell (eg, p-EIF2α, XBP1s, BIP, C/EBP homologous protein (CHOP), ATF3, and ATF6). (9) The T1D-associated gene TYK2 contributes to the activation of interferon alfa-mediated MHC1 expression in β-cells. (10) HLA-DR3/4 and HLA-DQ2/8 haplotypes of the HLA class II molecules of the MHC2 system are those associated with T1D. (11) Expression of MHC2 molecules by islet β-cells is an aberrant feature that confers β-cells the ability to bind β-Ag associated with T1D and engage CD4+ T-cells in an antigen-presenting cell (APC)-like manner, initiating autoimmunity. 2. Autoimmune processes (yellow numbers). Immune cell responses against antigens and β-cell–derived β-Ag play a central role in the pathogenesis of T1D. (1) Independently of MHC1 and MHC2 β-Ag presentation, β-cells under metabolic stress continuously secrete fused peptide fragments or hybrid insulin peptides (HIPs) that are recognized by APCs as autoantigen targets for pathogenic islet-infiltrating T-cells. (2) Some β-Ag are processed by APCs and presented to naïve CD4+ T-cells as antigens by MHC2 molecules on the surface of the APC. (3) CD4+ T-cell activation and differentiation depends on the signal strength received by the T-cell receptor (TCR) via the binding of MHC2 and on costimulation signals. CD28 and membrane-bound DPP4 (mDPP4) are prominent co-stimulatory molecules controlling the activation and behavior of naïve CD4+ T-cells. The mDPP4-mediated signal can be co-stimulated with APC in a caveolin-1-mDPP4-CD45-CD3-dependent manner activating the MAPK/ERK signaling pathway, or by the adenosine deaminase (ADA)-mDPP4 pathway activating proinflammatory NF-κB transcription factors. (4) CD4+ T-cells depend on the membrane action potential that is initiated by TCR stimulation for activation, intracellular Ca2+ homeostasis, cytokine production, and proliferation. Autoantigen-activated MHC2-TCR complex depolarizes the T-cell membrane and opens KV1.3 channels that are part of a signaling complex with P56lck (LCK), previously associated with impaired T-cell activation in T1D. K+ efflux opens Ca2+ release-activated channels (CRAC), resulting in significant Ca2+ influx and opening of KCA3.1 channels, thereby sustaining prolonged Ca2+ entry that is needed for further T-cell activation, cytokine release, and proliferation. (5) Upon TCR-activation and metabolic reprogramming, CD4+ T-cells also upregulate the expression of NMDAR, which are glutamate-activated and have an effect on cytokine production, T-cell proliferation, and differentiation. (6) In APCs, mDPP4-caveolin-1 interaction upregulates the expression of CD86 and subsequent engagement and recruitment of CD8+ cytotoxic T-cells, and cytokine secretion. The production by the APCs of cytokines such as IL-12 and interferon gamma promotes and accelerates further differentiation of the CD4+ T-cells into Th1-type cells and inhibits CD4+ Th2 cell production of IL-4 and IL-10. Additionally, interferon gamma upregulates the expression of NADPH oxidase/NOX family proteins that transport electrons from nicotinamide adenine dinucleotide and generate cytoplasmic reactive oxygen species (ROS). (7) Metabolic reprogramming of naïve CD4+ T-cells also leads to an increase in ROS and accelerated proliferation and activation of specialized immune cell subtypes, such as Th1, Th7, and Tfh. Activated CD4+ and CD8+ T-cells also secrete interferon gamma, IL-12, IL-18, and proinflammatory TNF-α and IL-1β, activating macrophages and stimulating the production of reactive nitrogen intermediates (RNIs). (8) Binding of CD8+ T-cells with β-cells, via a T-cell receptor (TCR)-MHC1 complex induces β-cell death through secretion of toxic molecules, such as perforin and granzymes. T-cells and macrophages can also destroy β-cells by secreting nitric oxide (NO°) and cytotoxic cytokines, subsequently activating a NF-κB signaling profile. Importantly, secretion of these cytokines by CD4+ T-cells may also increase expression of MHC1 on β-cells and promote direct assaults by CD8+ T-cells. (9) Autoimmune assaults on the β-cells by ROS, RNIs, actions of cytokines, and granzymes that activate caspase enzymes, lead to β-cell apoptosis and/or necrosis.

Fig. 3

T1D targets for therapeutic interventions in endocrine cells. β-cell: This figure illustrates the key targets for pharmaceutical intervention of oxidative stress, glucotoxicity, and apoptosis in pancreatic β-cells. (1) Thioredoxin-interacting protein (TxNIP) has an important role and the transcription is activated by carbohydrate-response element-binding protein (ChREBP) which binds to the TxNIP promotor and mediates glucose-induced TxNIP expression. During hyperglycemia, glucose is converted to Xu-5-P, with elevation of cytosolic Ca²⁺ ChREBP is translocated into the nucleus and translated into TxNIP. (2) First-generation Ca²⁺ blockers (diltiazem or verapamil) significantly reduce endogenous TxNIP mRNA expression due to the reduction of cytosolic Ca²⁺. The nuclear-cytoplasmic shuttling of ChREBP and binding to DNA are regulated by PKA- and AMPK-mediated phosphorylations. Metformin transiently inhibits complex I of the electron transport chain in the mitochondrion, leading to inhibition of V-ATPase and LKB1-mediated AMPK activation. Activated AMPK directly hinders ChREBP at Ser568, prevents cytosol-to-nuclear translocation, and inactivates its DNA-binding activity. (4) V-ATPase functions as a sensor switch between AMPK-induced catabolic metabolism and mTORC1 anabolic pathways. Activation of mTORC1 is a major increase of beta-cell mass by modulation of cyclin D2, D3, and Cdk4 activity. (5) Some PPI can also inhibit V-ATPase-Ragulator and induce AMPK-mediated response to ROS. Additionally, omeprazole preserves ALDH2 in mitochondria. Activation of ALDH2 in β-cells prevents apoptosis, enhances GSIS, and reduces both the mitochondrial and intracellular ROS levels. (6) Pharmaceutical interventions with GLP-1RA, GCGR mAbs, and DPP-4i-induced GLP-1 elevation stimulate GS-coupled ligands and initiate cAMP-PKA and cAMP-Epac signaling, thereby downregulating TxNIP expression levels in hyperglycemic conditions. (7) PKA phosphorylates ChREBP at Ser196, which inhibits nuclear import at Thr666, which inhibits DNA-binding activity. Downregulation of TxNIP expression reduces caspase-1 expression and prevents caspase-induced apoptosis. (8) β-catenin (β-Cat) is another pharmaceutical target that can promote proliferation, survival, and function of β-cells in diabetic conditions. GLP-1RA activation leads to β-Cat stabilization through PKA-mediated phosphorylation. (9) Additionally, Wnt-β-cat and PI3K-AKT-mTORC1 pathways share a common inhibition target GSK3β and independently can upregulate free cytosolic β-cat. (10) In the nucleus β-Cat forms the bipartite transcription factor β-cat/TCF with a TCF family member (eg, TCF7L2) upregulating GLP-1R and GIPR expression and several proliferative genes, including c-myc and cyclin D1. (11) PPI indirectly elevates serum gastrin. (12) Stimulation of G-coupled receptors, including gastrin receptor CCK2R, GABABR, G_i/o-GPCR, and insulin receptors activate PI3K-AKT pathway leading to activation of mTORC1 and inhibition/mediation of GSK3β, caspase-9 and Bcl-2-associated death promoter (Bad). (13) Expression of functional GABA_BR in human islets is restricted and tightly regulated by elevation in cAMP signaling. GABA_BR is primarily coupled to the G_i/o-GCGR and their activation leads to hyperpolarization induced closure of VGCC and insulin secretion. (14) NMDAR inhibition with dextromethorphan (DXM) significantly prolongs β-cell depolarization state promoting insulin secretion. (15) GCGR mAbs, such as IgG2 mAb volagidemab, bind to the human GCGR, and competitively block GCGR interaction with glucagon, thereby reducing cAMP and elevating AMPK. GCGR antagonism can improve glycemic control but cause adverse events in patients. (16) In β-cells activation of GABA_ARs with GABA, benzodiazepines, barbiturates, neurosteroids, and ethanol leads to Cl^- efflux and depolarization, opening of VGSC, VGCC, and promoting insulin release. α-cell: (1) Insulin inhibits glucagon secretion via insulin-dependent SGLT-2-induced stimulation of somatostatin release by δ cells, which downregulate cAMP, activate Gi/o-GPCR and hyperpolarize α-cell membrane. (2) Additionally, β-cells secretion of insulin and GABA inhibit glucagon secretion via IR activation of PI3K-AKT-dependent membrane assembly and activation of GABA_AR activity. (3) GABA_AR activation with GABA leads to Cl^- influx, α-cell membrane hyperpolarization, and suppression of glucagon secretion. (4) GABA and artemisinins can potentially reprogram α-cell into β-like-cell by repression or expression of key transcription factors. δ-cell: Somatostatin is a paracrine inhibitor of both insulin and glucagon. (1) The δ-cells are electrically excitable, and somatostatin secretion depends on SGLT2 activation by (2) insulin and K_ATP-induced depolarization, provided there are extracellular Na⁺ and elevated glucose levels. (3) SGLT2 inhibitors were seen to improve clinical parameters in T1D patients but can provoke euglycemic ketoacidosis and increase hepatic glucose production. Pancreatic exocrine cells: (1) Gastrin enhances ductal cell transdifferentiation into insulin-producing β-like cells. In ductal cells, concurrent administration of gastrin with epidermal growth factor or GLP-1RA has been shown to increase the β-cell mass and/or to improve glucose tolerance. (2) Exocrine cells are reprogrammed by upregulating gene expression of the endocrine progenitor markers including PDX1, and Nkx-6.1, and downregulating KRT20 expression present in mature duct cells. (3) In acinar cells, the antiapoptotic action of gastrin is mediated by PI3K-AKT, ERK, and MAPK signaling. The exact mechanism for transdifferentiation of pancreatic exocrine cells into beta-like cells has not been elucidated, but molecular interactions can include gastrin-induced upregulation of heparin-binding epidermal growth factor-like (HB-EGF), transactivation of EGFR, PI3K-AKT-mTORC1, and GLP-1-cAMP-PKA signaling cascades.

Fig. 4

T1D targets for therapeutic interventions in immune cells. Immune cells: (1) Anti-thymocyte globulin (ATG) induces broad nonspecific immunosuppression that is primarily mediated through the recognition of a series of antigens expressed on human lymphohematopoietic cells, such as CD2, CD3, CD4, and CD8 expressed on T-cells, CD19 and CD20 expressed on B cells, and CD11b, CD80, and CD86 expressed on antigen-presenting cells (APCs). ATG achieves immunosuppression by eliminating lymphocytes in the recirculating pool through complement-mediated intravascular lysis, apoptosis, and antigen-dependent cell-mediated cytotoxicity. Low-dose ATG reduced HbA1c 2 years after therapy in recent-onset T1D patients. (2) Anti-CD3 monoclonal antibody (teplizumab) blockade of CD3 receptors in T-cells induces a state of anergy in certain T-cell populations, making them unresponsive to specific stimuli, and promoting regulatory T-cell functions. In recent-onset T1D patients, teplizumab was observed to preserve β-cell function and slow down C-peptide degradation. Costimulatory signal blockade in T-cells: (3) Abatacept is a CTLA-4/Fc fusion protein that prevents T-cell CD28 interaction with its CD80/86 ligand on APCs, thereby limiting immune system activation. Abatacept modified progression of T1D by significantly impacting CD4+ cell subsets, thereby delaying the decline of C-peptide and improving HbA1c in recent-onset T1D patients. (4) Alefacept is an anti-CD2 fusion protein that has a dual function, as it triggers PCD of activated memory T-cells and inhibits the interaction between leukocyte-function-associated antigen (LFA-3) and CD2, effectively preventing costimulatory signaling for the activation and proliferation of T-cells. In recent-onset T1D patients, 2 12-week courses of alefacept delayed C-peptide decline and depleted CD4+, CD8+ T-cells, and effector memory T-cells for over a year after cessation of therapy. (5) High expression of membrane-bound dipeptidyl peptidase-4 (mDPP-4) is associated with the differentiation of T lymphocytes into Th1 (IL-2, interferon gamma) and Th17 (IL-6, IL-17, and IL-22) cells and upon activation of B cells. DPP-4 inhibitors (DPP-4i) disrupt the mDPP4-caveolin-1 nuclear factor kappa B (NFκB) activation pathway, which leads to a decrease in the expression of CD86 on antigen-presenting cells (APCs) and other monocytes. This limits the interaction between CD86 and CD28 on T-cells, resulting in a reduction in the proliferation and activation of antigen-specific T-cells. Additionally, DPP-4 inhibition prevents binding of mDPP-4 with adenosine deaminase (ADA) that would otherwise lead to the formation of a costimulatory CD3 signaling complex in CD4+ T-cells initiating CARMA-1 signaling. Furthermore, DPP-4 inhibitors prevent activation of Th1 cells, thereby leading to decreased secretion of proinflammatory cytokines: IL-1β, interferon gamma, TNF-α, and IL-2 from Th1 cells. GLP-1: (6) Activated T-cells express a higher number of functional glucagon-like peptide-1 receptors (GLP-1R) in human CD4+ T-cells and GLP-1R activation by GLP-1 receptor agonists (GLP-1RA) in Treg cells leads to increased IL-10 expression and enhanced cellular inhibitory function. T regulatory cells (Treg cells): (7) DPP-4 inhibition in CD4+ T-cells is found to promote the function of Treg cells and the production of the immunosuppressive cytokine TGF-β. Immunosuppressive functions of Treg cells are facilitated through CTLA-4 ligand binding to CD80/86 on APCs, expression of immunosuppressive cytokines: IL-10, TGF-beta and CD39-induced hydrolyzation of ATP to adenosine (ADO). Treg-derived ADO is a hydrolysis product of extracellular ATP cleaved in tandem by 2 Treg-associated ectonucleotidases, CD39 and CD73. (8) Most naïve CD8+ T-cells and a small number of mature CD4+ and Treg cells express CD73 on their surface. Enzyme-active CD73 is released from the CD8+ T-cell membrane upon activation, allowing Treg cell-driven ATP hydrolysis to occur, leading to adenosine formation. In immune cells, adenosine binds to the A2A receptor (A2aR), leading to the elevation of cAMP and interfering with the functions of activated T-cells and APCs, inhibiting their proliferation and cytokine production. IL-21: (9) IL-21 is produced primarily by CD4+ T-cells and is required for both Th17 cell differentiation and the generation of T follicular helper (Tfh) cells. IL-21 is the most prominent cytokine for the activation and differentiation of human B cells. IL-21 induces the differentiation of human naive and memory B cells into antibody-secreting plasma cells. Other cytokines, such as IL-4, greatly inhibit IL-21-driven plasma cell differentiation. IL-21 also directly regulates B-cell proliferation and apoptosis and can promote immunoglobulin production and isotype class switching. In addition, IL-21 signaling enhances the cytotoxicity of CD8+ T-cells and natural killer (NK) cells. A combination treatment of anti-IL-21 and GLP-1RA (liraglutide) preserved β-cell function in recent-onset T1D patients. This was demonstrated by a reduction in the concentration of C-peptide, as measured during a mixed-meal tolerance test (MMTT), from the baseline measurement to week 54 of treatment. B cells: (10) Anti-CD20 mAb (rituximab) effectively depletes mature B cells by various mechanisms inducing cell death, including DNA fragmentation, complement-dependent cytotoxicity (CDC), and programmed cell death (PCD). CD20 is reported to regulate B-cell differentiation and growth as well as adjusting Ca2+ transport. Notably, CD20 is detectable in pre-B cells to mature B cells but is absent in antigen-producing plasma cells. GABA: (11) Gamma-aminobutyric acid (GABA) has broad immune-modulating properties. It controls the release of cytokines from CD4+ T-cells and anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs). Importantly, GABA suppresses the release of 47 cytokines in PBMCs from T1D patients and regulates pro- and anti-inflammatory cytokine production in a concentration-dependent manner. Engagement of the GABAA receptor (GABAAR) induces depolarization of the membrane potential, leading to inhibition of T-cell responses. B cells secrete GABA, which then inhibits inflammatory cytokine production in CD8+ T-cells and stimulates monocyte differentiation into IL-10-secreting immunosuppressive cells. Calcium blockade: (12) Lymphocyte calcium channel blockade may be an effective immunosuppressive strategy. Verapamil had a significant impact on T-cell activation by strongly inhibiting the expression of CD25 (which is typically present in Tregs), CD40L, and CD69. This inhibition is likely due to the failure of Ca2+-dependent transcription factors to activate gene transcription. Nuclear factor of activated T-cells (NFAT) is triggered by Ca2+, which also triggers the production of other transcription factors, including IRF4 and HIF-1α, that control the metabolic switch, cell cycle progression, and proliferation of activated human T-cells. Verapamil partially preserves β-cell function, as shown by C-peptide secretion in children and adolescents with recent-onset T1D. Proton pump inhibitors: (13) In immune cells, proton pump inhibitors (PPIs) suppress T-cell responses by decreasing the expression of the T-cell receptor (TCR)-activated membrane zinc transporter Zip8, thereby lowering the cytoplasm-free zinc (Zn) concentration. PPI-induced decrease in Zip8 expression increases transcription factor CREMα, which dramatically downregulates IL-2 production, while decreases in the transcription factor pCREB downregulates production of interferon gamma in lymphocytes. In monocytes, PPIs were found to reduce the production of several inflammatory cytokines: TNF-α, IL-1β, IL-6, and NFκB. Moreover, PPIs inhibit the activation of neutrophils and monocytes and deplete intracellular and extracellular neutrophil reactive oxygen species (ROS) and nitric oxide (NO). PPI with DPP-4i in recent-onset T1D patients did not achieve C-peptide preservation, but due to high safety, PPI and DPP-4i have been suggested to be used in combination with other drugs. Preliminary results show that a combination of GABA, DPP-4i, and PPI as an adjunct to insulin therapy improves glycemic control in patients with T1D and elevates C-peptide levels in recent-onset T1D patients. Beta-cells: Cytokine blockade. (A) During the progression of T1D macrophages and T-cells invade the islets and secrete proinflammatory cytokines. The combination of TNF-α and interferon gamma synergistically induces β-cell apoptosis through activation of JNK/SAPK, resulting in the production of reactive oxidative species (ROS) and loss of mitochondrial transmembrane potential (ΔΨm). Proinflammatory cytokine blockade may act to prevent deleterious effects on β-cell survival and function in the islet microenvironment. Anti-inflammation and cytokine-modifying therapies showed varying degrees of effectiveness as TNF-α monoclonal antibodies (eg, Golimumab and Etanercept) postponed C-peptide loss in patients with recent-onset T1D. Canakinumab binds human IL-1β with high affinity and neutralizes its biological activity while Anakinra is an IL-1 receptor antagonist. Due to high safety, but insufficient efficacy in recent-onset T1D patients, canakinumab and anakinra have been suggested for IL-1β blockade as part of combination therapies. Verapamil + IGF-1: (B) Verapamil downregulates Ca2+ influx and thereby disrupts the formation of thioredoxin-interacting protein (TxNIP), reduces β-cell expression of IGF-binding protein 3 (IGFBP3) and thereby elevates IGF-1 induced signaling via increased IGF-1. (C) Stimulation of IGF-1R initiates PI3K/Akt signaling, which enables the activation of mTORC1. Upon activation mTORC1 phosphorylates the 4EBP1 protein, promoting cell growth, and the p70 ribosomal protein S6 kinase (S6K1), resulting in enhanced ribosomal biogenesis, mitochondrial biogenesis, and oxygen consumption. PI3K/Akt signaling also promotes β-cell-, but inhibits α-cell-related gene expression, as well as inhibiting β-cell apoptosis in the context of inflammatory cytokines and oxidative stress. Importantly, insulin receptor (IR) and IGF-1R are highly homologous and share PI3K/Akt and Ras/MAPK signaling pathways IR largely controls metabolism, whereas IGF-1R controls growth. (D) IGFBP-3 is a negative regulator of β-cell mass independent of IGF-1, which is a positive regulator. IGFBP-3 is a binding ligand to the death receptor TMEM219, which is widely expressed in islet β-cells. Bound TMEM219 triggers Caspase-8-mediated apoptosis of β-cells. (E) A 26-week course of a small tyrosine kinase inhibitor, imatinib mesylate (Gleevec, STI571) preserved β-cell function at 12 months in adults with recent-onset T1D. Imatinib acts as a β-cell protective drug, as it reduces ER stress and consequent β-cell apoptosis by inhibiting ABL kinase binding and hyperactivation of ER transmembrane kinase’s endoribonuclease (IRE1α RNase).